Diagnostic sensing apparatus

ABSTRACT

A sensing apparatus and methods for measuring or detecting an analyte present in a biological system are provided. The methods entail use of the sensing apparatus that contains a reporter system specific for the analyte of interest, where the reporter system is either affixed to a planar backing or attached to particles that are delivered to the superficial layers of the skin. The reporter system includes a reporting reagent that absorbs or emits a detectable radiation and is placed in communication with the analyte, or in communication with tissue or body fluids suspected of containing the analyte. The sensing apparatus is illuminated, and a radiation signal from the reporting reagent is measured or detected and then associated with the presence or quantity of analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not Applicable

FIELD OF THE INVENTION

The invention relates generally to an apparatus and methods ofmonitoring the presence and/or concentration of a target analyte presentin an aqueous biological system. More particularly, the inventionrelates to an apparatus and methods for determining the presence ormeasuring the concentration of one or more analytes in a transdermallyaccessed sample. One important application of the invention involves anapparatus and method for monitoring blood glucose using non-invasive orminimally invasive monitoring techniques.

BACKGROUND OF THE INVENTION

This application relates to an apparatus and methods for detecting andquantifying analytes in body fluids using fluorescence techniques.

Various types of apparatus are currently used to measure analytes. Theyinclude pads, membrane dipsticks, swabs, tubes, vials, cuvettes, andcapillaries. Reagents for determining the presence or concentration ofspecific analytes may be present in or added to these devices to measurethe analyte of interest. For example, dipsticks containing reagents,that measure hormones, are useful in determining whether the user ispregnant.

Numerous methods for detecting and quantifying analytes in body fluidsare known. These tests typically rely on physiological fluid samplesremoved from a subject, either using a syringe or by pricking the skin.For example, in the case of glucose these methods include variouscolorimetric reactions, measuring a spectrophotometric change in theproperty of any number of products in a glycolytic cascade or measuringthe oxidation of glucose using a polarimetric glucose sensor.

Diabetes is a major health concern, and treatment of the more severeform of the condition, Type I (insulin-dependent) diabetes, requires oneor more insulin injections per day. Insulin controls utilization ofglucose or sugar in the blood and prevents hyperglycemia, which, if leftuncorrected, can lead to ketosis. On the other hand, improperadministration of insulin therapy can result in hypoglycemic episodes,which can cause coma and death. Hyperglycemia in diabetics has beencorrelated with several long-term effects, such as heart disease,atherosclerosis, blindness, stroke, hypertension and kidney failure.

The value of frequent monitoring of blood glucose as a means to avoid orat least minimize the complications of Type I diabetes is wellestablished. According to the National Institutes of Health, glucosemonitoring is recommended 4-6 times a day. Patients with Type I(non-insulin-dependent) diabetes can also benefit from blood glucosemonitoring in the control of their condition by way of diet andexercise.

Conventional blood glucose monitoring methods generally require thedrawing of a blood sample (e.g., by finger prick) for each test, and adetermination of the glucose level using an instrument that readsglucose concentrations by electrochemical or colorimetric methods. TypeI diabetics must obtain several finger prick blood glucose measurementseach day in order to maintain tight glycemic control. However, the painand inconvenience associated with this blood sampling has lead to poorpatient compliance, despite strong evidence that tight controldramatically reduces long-term diabetic complications. In fact, theseconsiderations can often lead to an abatement of the monitoring processby the diabetic.

To satisfy the need for simpler and less painful sensing and monitoringneeds of the population, this invention provides for a simple sensingapparatus and methods of monitoring for the presence of analytes in bodyfluids. The methods are non-invasive or minimally invasive and havelittle or no pain associated with the monitoring steps helping toincrease patient compliance.

BRIEF SUMMARY OF THE INVENTION

The present invention provides apparatus and methods for sampling ananalyte present in a biological system. Accordingly, it is a primaryobject of the present invention to provide a sensing apparatus. Theapparatus comprises a substantially planar occlusive backing and areporter system. The reporter system absorbs or emits a detectableradiation, and is attached, coupled, adhered, or otherwise connected toa first planar surface of the occlusive backing. The reporter systembinds an analyte of interest, and the ability of the reporter system toabsorb or emit radiation is detectably altered in aconcentration-dependent manner when the analyte is bound to the reportersystem. It is preferable that the reporter system is attached to anocclusive backing that has sufficient drape characteristics to allow forpositioning of the apparatus over an uneven skin or mucosal surface,particularly allowing the apparatus to be positioned over a surface on alimb or other body part and remain in place despite normal bodilymovements and/or physical changes (e.g., perspiration) affecting thesurface.

In one aspect of the invention, the reporter system comprises a specificbinding pair having a first component that is an analyte-specificbinding ligand comprising a first light-absorbing material, and a secondcomponent that binds to the binding ligand of said first component andcomprises a second light-absorbing material. Binding of the secondcomponent to the first component is reversible, and the analyte binds tothe first component in a competitive manner, thereby displacing thesecond component. In turn, displacement of the second component producesa detectable alteration in the energy transfer between the firstcomponent and the second component, wherein such alteration isproportional to the concentration or amount of said analyte that bindsto the first component. In certain embodiments, the binding ligand canbe a glucose binding ligand, and the analyte of interest is glucose.More particularly, the glucose binding ligand can be concanavalin-A, andthe second component of the reporter system can comprise a dextranglycoconjugate. The detectable alteration in the energy transfer betweenthe first component and the second component can comprise anon-radiative fluorescence resonance energy transfer between the firstand second light-absorbing materials, and in certain preferredembodiments, the first component of the specific binding pair istetramethylrhodamine isothiocyanate-concanavalin A (“TRITC-ConA”) andthe second component of the specific binding pair is fluoresceinisothiocyanate-dextran (“FITC-dextran”).

It is also a primary object of the present invention to provide a methodfor detecting the presence or amount of an analyte present beneath atarget skin or mucosal surface of an individual. The method entails: (a)disrupting the target surface to create one or more passages in thatsurface sufficient to allow said analyte to flow, exude, diffuse orotherwise pass from beneath the target surface to the target surface;(b) placing a sensing apparatus constructed according to the presentinvention in contact with the target surface; and (c) detecting analteration in the ability of the reporter system to absorb or emitradiation, thereby obtaining a signal indicative of the presence and/oramount of analyte present beneath the target surface.

In certain aspects of the invention, the target surface is disrupted byaccelerating-small particles into the target-surface.-Such particlestypically have a size ranging from about 0.1 to 250 microns (nominaldiameter). In certain preferred embodiments, the particles have a sizeranging from about 10 to 70 microns. It is also preferred that theanalyte of interest is glucose.

It is yet another primary object of the present invention to provide amethod for quantifying glucose present in a body fluid beneath a targetsurface. The method entails: (a) accelerating particles into the targetsurface, wherein acceleration of such particles into the target surfaceis effective to allow passage of glucose from beneath the target surfaceto the target surface; (b) contacting the glucose present at the targetsurface with a specific binding pair comprising a first component whichis a glucose binding ligand containing a first light-absorbing material,and a second component which is a glycoconjugate containing a secondlight-absorbing material. The excited state energy level of the firstlight-absorbing material overlaps with the excited state energy level ofthe second light-absorbing material, and the ligand and glycoconjugatepair is chosen such that they reversibly bind to each other therebyallowing glucose present at the target surface to displace theglycoconjugate and competitively bind to the ligand; (c) determining theextent to which non-radiative fluorescence resonance energy transferoccurs between the first light-absorbing and the second light-absorbingmaterial in the presence of the glycoconjugate displaced by glucose andthe ligand reversibly bound to glucose; and (d) comparing the result ofstep (c) with the relationship between the extent of non-radiativeenergy transfer between the first light-absorbing material and thesecond light-absorbing material and glucose concentration in the bodyfluid determined in a calibration step.

In the practice of the method, acceleration of the particles into thetarget surface serves to permeabilize the target surface. In certainaspects, the particles are accelerated toward the target surface using aparticle injection device (needleless syringe).

It is also a primary object of the invention to provide a method fordetecting the presence or amount of an analyte present beneath a targetskin surface of an individual. The method entails: (a) providing aparticulate reporter system, wherein the reporter system binds theanalyte of interest and the ability of said reporter system to absorb oremit radiation is altered in a concentration-dependent manner when theanalyte is bound to the reporter system, and the particulate reportersystem is comprised of a homogenous population of particles each havinga size ranging from 0.1-250 microns; (b) administering the reportersystem into the target skin surface such that the particulate reportersystem is delivered to a substantially uniform and homogenous depthwithin the skin; (c) allowing the reporter system to contact theanalyte; and (d) detecting an alteration in the ability of the reportersystem to absorb or emit radiation thereby obtaining a signal indicativeof the presence or amount of analyte present beneath the target skinsurface.

It is preferred that the particulate reporter system is delivered usinga particle injection device (needleless syringe), and that the particlesare delivered at a depth of about 1-50 microns below the target surface.Although a number of particle sizes will be suitable for use in themethod, it is preferable that the particles are provided in a homogenoussize, and that they have a size ranging from about 10 to 70 microns.

In one aspect, the method is practiced using a reporter system thatcomprises a specific binding pair having a first component that is ananalyte-specific binding ligand and includes a first light-absorbingmaterial, and a second component that binds to the binding ligand of thefirst component and includes a second light-absorbing material. Thebinding of the second component to the first component is reversible,and the analyte binds to the first component in a competitive manner,thereby displacing the second component In turn, the displacement of thesecond component produces a detectable alteration in the displacement ofthe second component and produces a detectable alteration in the energytransfer between the first component and the second component, whereinsuch alteration is proportional to the concentration or amount of theanalyte that binds to the first component In certain embodiments, thebinding ligand can be a glucose binding ligand, and the analyte ofinterest is glucose. More particularly, the glucose binding ligand canbe concanavalin-A, and the second component of the reporter system cancomprise a dextran glycoconjugate. The detectable alteration in theenergy transfer between the first component and the second component cancomprise a non-radiative fluorescence resonance energy transfer betweenthe first and second light-absorbing materials, and in certain preferredembodiments, the first component of the specific binding pair istetramethylrhodamine isothiocyanate-concanavalin A (“TRITC-ConA”) andthe second component of the specific binding pair is fluoresceinisothiocyanate-dextran (“FITC-dextran”). These components arefluorophore labeled ligands formed by reaction of the parent lectin,concanavalin-A and a glycoconjugated dextran with the respective dyesreactive by virtue of having isothiocyanate moieties.

In the above-described methods, the analyte can be any specificsubstance or component that one-is desirous of detecting and/ormeasuring in a chemical, physical, enzymatic, or optical analysis. Suchanalytes include, but are not limited to, toxins, contaminants, aminoacids, enzyme substrates or products indicating a disease state orcondition, other markers of disease states or conditions, drugs ofrecreation and/or abuse, performance-enhancing agents, therapeuticand/or pharmacologic agents, electrolytes, physiological analytes ofinterest (e.g., calcium, potassium, sodium, chloride, bicarbonate (CO₂),glucose, urea (blood urea nitrogen), lactate, and hemoglobin), lipids,and the like. In preferred embodiments, the analyte is a physiologicalanalyte of interest, for example glucose, or a chemical that has aphysiological action, for example a drug or pharmacological agent. Aswill be understood by the ordinarily skilled artisan upon reading thepresent specification, there are a large number of analytes that can besampled using the present invention.

An advantage of the invention is that the instant sampling processes canbe readily practiced inside and outside of the clinical setting andwithout pain.

These and other objects, aspects, embodiments and advantages of thepresent invention will readily occur to those of ordinary skill in theart in view of the disclosure herein.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified analytes or process parameters as such may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyand for all purposes.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although a number of methodsand materials similar or equivalent to those described herein can beused in the practice of the present invention, the preferred materialsand methods are described herein.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a particle” includes a mixture of two or more suchparticles, reference to “an analyte” includes mixtures of two or moresuch analytes, and the like.

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The term “analyte” is used herein in its broadest sense to denote anyspecific substance or component that is being detected and/or measuredin a physical, chemical, biochemical, electrochemical, photochemical,spectrophotometric, polarimetric, colorimetric, or radiometric analysis.A detectable signal can be obtained, either directly or indirectly, fromsuch a material. In preferred embodiments, the analyte is aphysiological analyte of interest (e.g., a physiologically activematerial), for example glucose, or a chemical that has a physiologicalaction, for example a drug or pharmacological agent. Examples includematerials for blood chemistries (blood pH, pO₂, pCO₂, Na⁺, Ca⁺⁺, K⁺,lactic acid, glucose, and the like), for hematology (hormones, hormonereleasing factors, coagulation factors, binding proteins, acylated,glycosylated, or otherwise modified proteins and the like), andimmuno-diagnostics, toxins, contaminants, amino acids, enzymes, enzymesubstrates or products indicating a disease state or condition,immunological substances, other markers of disease states or conditions,performance-enhancing agents, therapeutic and/or pharmacologic agents,electrolytes, physiological analytes of interest (e.g., calcium,potassium, sodium, chloride, bicarbonate ([HCO₂]⁻²), glucose, urea(blood urea nitrogen), lactate, and hemoglobin), materials for DNAtesting, nucleic acids, proteins, carbohydrates, lipids, electrolytes,metabolites (including but not limited to ketone bodies such as3-hydroxybutyric acid, acetone, and acetoacetic acid), therapeutic orprophylactic drugs, gases, compounds, elements, ions, drugs ofrecreation and/or abuse, anabolic, catabolic or reproductive hormones,anticonvulsant drugs, antipsychotic drugs, alcohol cocaine,cannabinoids, opiates, stimulants, depressants, and their metabolites,degradation products and/or conjugates. The term “target analyte” refersto the analyte of interest in a specific monitoring method or technique.

The term “analogue” refers to a material that has at least some bindingproperties in common with those of the analyte such that there areligands that bind to both. The analogue and the analyte, however, do notbind to each other. The analogue may be a derivative of the analyte suchas a compound prepared by introducing functional chemical groups ontothe analyte that do not affect at least some of the binding propertiesof the analyte. Another example of a derivative is a lower molecularweight version of the analyte that nonetheless retains at least some ofthe binding properties of the analyte.

As used herein, the term “pharmacological agent” intends any compound orcomposition of matter which, when administered to an organism (human oranimal subject), induces a desired pharmacologic and/or physiologiceffect by local and/or systemic action.

As used herein, the term “sampling” means access to and monitoring of asubstance from any biological system from the outside, e.g., across amembrane such as skin or tissue. The membrane can be natural orartificial, and is generally animal in nature, such as natural orartificial skin, blood vessel tissue, intestinal tissue, and the like. A“biological system” thus includes both living and artificiallymaintained systems.

The term “individual” is used interchangeable herein with the term“subject,” and encompasses any warm-blooded animal, particularlyincluding a member of the class Mammalia such as, without limitation,humans and nonhuman primates such as chimpanzees and other apes andmonkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult, child andnewborn subjects, whether male or female, as well as fetuses, areintended to be covered.

The term “sensing apparatus” encompasses any device that can be used tomeasure the concentration of an analyte of interest. A preferred sensingapparatus will be a substantially planar backing with a reporter systemconnected to it. The reporter system will measure the level of ananalyte present in a body fluid. Preferred analytes will be found ininterstitial fluid. Detection and/or quantification of a radiationsignal can be carried out using readily available radiationemission/adsorption monitoring devices. Examples of fluorogenic systemsinclude non-radiative energy transfer systems.

The term “non-radiative fluorescence resonance energy transfer” is usedinterchangeably with the acronym “FRET” herein. The process involves atransfer of energy from a first fluorescent reagent that acts as anenergy donor to a second fluorescent reagent that acts as an energyacceptor.

The term “fluorescence reagent” is used interchangeably with the term“reporter system” and refers to a material whose fluorescence behavior(e.g., intensity, emission spectrum, or excitation spectrum) changes inthe presence of the analyte being detected In some embodiments, thefluorescent reagent binds reversibly to the analyte. For example, thereagent may be a fluorophore, or a compound labeled with a fluorophore,that binds directly to the analyte. It is the fluorescence behavior ofthis molecule (or compound labeled with this molecule) that changes as aresult of analyte binding.

The reagent may also include more than one component. For example, itmay include an analogue to the analyte labeled with a fluorophore and aligand (e.g., an antibody, receptor for the analyte, lectin, enzyme, orlipoprotein) that binds competitively (and specifically) to the analogueand the analyte. In this case, it is the fluorescence behavior of thelabeled analogue that changes as a result of ligand binding to analyte.Conversely, the ligand may be labeled, rather than the analogue, inwhich case it is the fluorescence behavior of the labeled ligand thatchanges.

The reagent may also include two components, one of which is labeledwith an energy-absorbing donor molecule and the other of which islabeled with an energy-absorbing acceptor molecule; the donor andacceptor have overlapping excited state energy levels. One or bothmolecules forming the donor-acceptor pair can be fluorophores.Regardless, however, it is the fluorescence associated with thenon-radiative resonance energy transfer from donor to acceptor that ismeasured. The components may be members of a specific binding pair(e.g., an analogue of the analyte and a ligand capable of bindingcompetitively (and specifically) to both the analogue and the analyte)or ligands (e.g., antibodies or oligonucleotides) that bind specificallyto different portions of the analyte.

FREE can also be measured where a single reagent capable of binding tothe analyte is labeled with both donor and acceptor molecules.

The term “fluorophore” refers to a molecule that absorbs energy andemits light

The term “fluorescence” refers to radiation emitted in response toexcitation by radiation of a particular wavelength. It includesshort-lived (nanosecond range) and long-lived excited state lifetimes,the latter is sometimes referred to as phosphorescence.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a sensing apparatus and methods for samplinganalytes present in a biological system, typically a physiologicallyactive material that is present beneath a target skin or mucosal surfaceof an individual.

An ideal sensing apparatus should contain a reporting system and becapable of detecting a wide range of physiological concentrations ofanalyte. As used herein, “physiological concentration” refers to theconcentration of analyte found in both normal and pathological states.For example, in the case of glucose it refers to glucose levels found innormal, hypoglycemic, and hyperglycemic patients. In the case ofanalytes not normally present in the biological system, the reportingsystem should be capable of detecting trace amounts of the substance.

The sensing apparatus should also be reliable, reusable and easy to use.In addition, the sensing apparatus should be non-invasive or minimallyinvasive.

The sensing apparatus can be constructed from a wide range of materials,including both rigid and pliable materials. Preferably, the apparatus isconstructed of a planar material that is pliable such that it can moldto the surface to which it is applied. In one embodiment, the planarmaterial is transparent so that light can be transmitted through thematerial from an external light source and light can be detected frombeneath the material by an external detector. Ideally the light sourceand detector would be in a single unit.

sensing apparatus is preferably made out of flexible material that isimpervious to moisture. Such materials can include but not be limited toplastic or polymeric materials including thermoplastics such aspolycarbonates, polyesthers (e.g., MYLAR™ and polyethylene terephthalate(PET)), polyvinyl chloride (PVC), polyethylene glycol hydrogel (PEGH),polyurethanes, polyethers, polyamides, polyimides, or copolymers ofthese thermoplastics, such as PETG (glycol-modified polyethyleneterephthalate). Other suitable flexible, water impervious materials arewell known to those of skill in the art The material can be cut in avariety of shapes and sizes as required for the location that thesensing apparatus will be used and the volume of body fluid required forsampling the analyte of interest. The planar material will typicallyrange from 0.01 up to 2 or more millimeters in thickness, depending uponthe material used.

The sensing apparatus will have an adhesive component to at least oneedge, or portion of an edge of the surface that comes in contact withthe target skin or mucosal surface of an individual. Preferably theentire edge of the surface of the sensing apparatus that comes incontact with the target skin or mucosal surface will have an adhesivecomponent for adhering the sensing apparatus to the individual.Alternatively, the entire surface of the sensing apparatus that comes incontact with the target skin or mucosal surface of the individual willbe covered with the adhesive material. Typically the adhesive will be apressure-sensitive adhesive. Pressure-sensitive adhesives generallycomprise a pressure-sensitive adhesive component, a tackifier andsoftener. Examples of such pressure sensitive adhesive componentsinclude but are not limited to natural and synthetic resins such asnatural rubber, polyisobutylene rubber, polybutadiene rubber, siliconerubber, polyisoprene rubber, styrene-isopropylenestyrene block copolymer(abbreviated “SIS”) and acrylate copolymer, which are used either aloneor as a mixture of two or more of them. The content of thepressure-sensitive adhesive component(s) of the pressure sensitiveadhesive may range from 10-50% by weight, preferably from 15-45% byweight, still more preferably 20-40% by weight.

The tackifier used for adjusting the pressure-sensitive adhesivenessincludes rosin, hydrogenated rosin, and esters thereof, polyterpeneresin, petroleum resin, and ester gum, etc. which are used either aloneor as a mixture of two or more of them. The content of the tackifier(s)in the pressure-sensitive adhesive may be up to 40% by weight, andpreferably in the range from 5 to 35% by weight, still more preferablyfrom 15 to 30% by weight.

Further, the softener to be used in the present invention may be one ormore members selected from among liquid paraffin, polybutene, liquidpolyisobutylene and animal and vegetable oils. The content of thesoftener(s) in the pressure sensitive adhesive may range from 5 to 60%by weight, preferably from 10 to 50% by weight, still more preferablyfrom 25 to 45% by weight.

If necessary, the pressure-sensitive adhesive may further contain one ormore fillers selected from among titanium dioxide, synthetic aluminumsilicate, zinc oxide, calcium carbonate, starch acrylate, silica and soforth. The content of the filler(s) in the pressure-sensitive adhesivemay be up to 5% by weight, and preferably ranges from 0.1 to 4% byweight, still more preferably 1 to 3% by weight.

The sensing apparatus will contain a reporter system for measuring thepresence of specific analytes. Suitable reporter systems and analytesare described herein. Generally, the reporter systems will containmultiple components for detecting and/or measuring the presence of theanalyte of interest. One component of the reporter system will beattached, adhered or otherwise fixably connected to the planar backingof the sensing apparatus. Preferably, this component can be a ligandthat binds the analyte of interest. Alternatively, the components of thereporter system can be contained in a porous matrix that is attached tothe planar occlusive backing of the sensing apparatus. The porous matrixmay be attached to the occlusive backing using the pressure-sensitiveadhesive described supra, or other adhesive well know to those of skillin the art.

The porous matrix may be composed of liquid permeable material,including but not limited to cellulose derivatives such as cellulose,carboxymethylcellulose, carboxymethylcellulose salts,hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose,ethylcellulose, carboxymethylethylcellulose,hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, celluloseacetate, cellulose nitrate, cellulose acetate phthalate andhydroxypropymethylcellulose phthalate; porous gels such aspoly-2-hydroxyethyl methacrylate, polyacrylate, polyacrylic acid andpolyvinyl alcohol-polyacrylic acid composite; fibrous matrixes such aspolyurethane, polyester, polyethylene, polyvinyl chloride,polyvinylidene fluoride and nylon; papers (such as nonwoven paper andfilter paper); cloths (such as staple fiber, cotton, silk and syntheticfibers); and porous ceramics such as silica, alumina, titania, zirconia,and ceria, which may be used either alone, or as a mixture of two ormore of them. The pore of the porous matrix will be of such a size as toallow the ingress and egress of the fluid sample while retaining thecomponents of the reporter system. Preferably, the porous matrix may bein particulate form. Still preferably, the particles will have a size inthe range of 0.1 to 250 Sun and more preferably in the range of 10 to 70μm.

The sensing apparatus may be applied to the target surface andsubsequently contacted with a detection means other than those describedherein to detect the analyte. The sensing apparatus may comprise ahydrogel. Suitable gelling agents for forming a hydrogel include agar,modified starches, amylopectin, carbopol, calcium, calcium lactate,cellulose gum, klucel (HPMC), natrosol, gelatin powder or sodiumalginate. The gelling agents may be present in water at levels such as 1to 4% by weight in water.

Alternatively, a hydrogel may be applied to the target surface andsufficient time allowed for analyte from the target surface toequilibrate in the gel prior to the detection step. The time may bequite short such as from 30 seconds to 5 minutes. Detection may then becarried out by applying the sensing apparatus to the gel. Alternatively,hydrogels containing analyte-specific reporter systems can be preparedby readily available techniques familiar to the ordinarily skilledartisan and used as described supra.

The invention also provides in vivo methods for detecting an analyte inan individual (as used herein, “detecting” may include qualitativelydetermining the presence of an analyte, as well as quantitativelymeasuring its concentration). The reporter system is placed incommunication with sampled analyte or in contact with tissue or bodyfluids (e.g., interstitial fluid) of the individual suspected ofcontaining the analyte. Such placement can be considered as permittingnon-invasive or minimally invasive detection and monitoring of theanalyte. The reporter system includes a fluorescence reagent fordetecting the analyte. Once the reporter system is in place, it isilluminated with radiation transdermally and the fluorescence from thefluorescence reagent associated with the presence of the analyte ismeasured.

In the practice of the present invention, the in vivo methods generallyentail two steps, a sampling (accessing) step and a detection step. Thesampling, or “accessing” step can be generalized as follows. A targetsurface is selected and cleaned with a suitable solvent The targetsurface is then disrupted in some manner sufficient to createmicro-passages that allow access to a quantity of an analyte. In thisregard, the analyte may be present in a fluid that flows, exudes,diffuses, perfuses, or otherwise passes from beneath the target surface,through the micro-passages to the target surface. In a preferredembodiment small sampling particles are accelerated into and/or across atarget surface. These sampling particles are accelerated to a speedsufficient to penetrate the skin or mucosal layer at the target site,thereby breaching the natural barrier function of the skin or mucosaltissue and allowing access to an analyte present beneath the targetsurface. The target surface generally has an overall size ranging fromabout 0.1 to about 5 cm².

The sampling particles typically comprise an inert material. Thematerial may be dissolvable such as commonly employed physiologicallyacceptable soluble materials including sugars (e.g., mannitol, sucrose,lactose, trehalose, and the like) and soluble or dissolvable polymers,e.g., swellable natural gels such as agarose. Alternatively, thesampling particles can be comprised of insoluble materials such asstarch, TiO₂, calcium carbonate, phosphate salts, hydroxy-apatite, oreven synthetic polymers or metals such as gold, platinum or tungsten.Insoluble materials are sloughed off with the normal skin or mucosaltissue renewal process. Preferred materials are lactose, mannitol andpolyethylene glycol, such as PEG 8000.

If desired, the sampling particles can be coated with a locally activeagent that facilitates the sampling step. For example, the samplingparticles can be coated with or contain a pharmacological agent such asa vasoactive agent or an anti-inflammatory agent. The vasoactive agentis generally used to provide short-acting vasoactivity (e.g., up to 24hours) in order to maximize, hasten or prolong fluid access (optimizeanalyte access), whereas the anti-inflammatory agent is generally usedto provide local anti-inflammatory action to protect the target site.The sampling particles can also be coated with or contain an osmoticallyactive agent to facilitate the sampling process;

The sampling particles can be delivered from a particle injectiondevice, e.g., a needleless syringe system as described in commonly ownedInternational Publication Nos. WO 94/24263, WO 96/04947, WO 96/12513,and WO 96/20022, all of which are incorporated herein by reference.Delivery of sampling particles from these needleless syringe systems isgenerally practiced with particles having an approximate size generallyranging from 0.1 to 250 μm preferably ranging from about 10-70 μm.Particles larger than about 250 μm can also be delivered from thedevices, with the upper limitation being the point at which the size ofthe particles would cause untoward pain and/or damage to the tissue.

The actual distance to which the delivered particles will penetrate atarget surface depends upon particle size (e.g., the nominal particlediameter assuming a roughly spherical particle geometry), particledensity, the initial velocity at which the particle impacts the surface,and the density and kinematic viscosity of the targeted skin tissue. Inthis regard, optimal particle densities for use in needleless injectiongenerally range between about 0.1 and 25 g/cm³, preferably between about0.9 and 1.5 g/cm³, and injection velocities generally range betweenabout 100 and 3,000 m/sec. With appropriate gas pressure, particleshaving an average-diameter of 10-70 μm can be readily acceleratedthrough the nozzle at velocities approaching the supersonic speeds of adriving gas flow. Preferably, the pressure used when accelerating theparticles will be less than 30 bar, preferably less than 25 bar and mostpreferably 20 bar or less.

Alternatively, the sampling particles can be delivered from aparticle-mediated delivery device such as a so-called “gene-gun” typedevice that delivers particles using either a gaseous or electricdischarge. An example of a gaseous discharge device is described in U.S.Pat. No. 5,204,253. An explosive-type device is described in U.S. Pat.No. 4,945,050. One example of a helium discharge-type particleacceleration apparatus is the PowderJect XR® instrument (PowderJectVaccines, Inc., Madison, Wis.), which instrument is described in U.S.Pat. No. 5,120,657. An electric discharge apparatus suitable for useherein is described in U.S. Pat. No. 5,149,655. The disclosure of all ofthese patents is incorporated herein by reference.

Other methods for disrupting the target surface, in a way thatmicro-pathways are formed in a target skin or mucosal surface to provideaccess to analyte beneath the target surface, are well known in the art.The term “micro-pathways” refers to microscopic perforations and/orchannels in the skin caused by pressure (water or particle injection),mechanical (micro lancets), electrical (thermal ablation,electro-poration, or electroosmosis), optical (laser ablation), andchemical methods or a combination thereof. For example, U.S. Pat No.5,885,211 describes five specific techniques for creating micro-pathwayswhich entail: ablating the surface with a heat source such that tissuebound water is vaporized; puncturing the surface with a microlancetcalibrated to form a micropore; ablating the surface by focusing atightly focused beam of sonic energy; hydraulically puncturing thesurface with a high pressure jet of fluid; and puncturing the surfacewith short pulses of electricity to form a micro-pathway. Anotherspecific technique is described in U.S. Pat. Nos. 6,219,574 and6,230,051, which describe a device having a plurality of microblades.The microblades are angled and have a width of 10 to 500 microns and athickness of 7 to 100 microns and are used to provide superficialdisruptions in a skin surface.

Disruption of the target surface allows access to the analyte ofinterest that was otherwise not accessible at the target surface. Forexample, disruption of the target surface can produce micro-pathwaysthat allow a small amount of a fluid sample (e.g., a body fluid) toflow, exude or otherwise pass to the target-surface via mass fluidtransport, wherein the fluid contains the analyte of interest. The term“body fluid” refers to biological fluid including, but not limited tointerstitial fluid, blood, lymph, sweat, or any other body fluidaccessible at the surface of suitably disrupted tissue. The term “massfluid transport” refers to the movement of fluids, such as body fluid.This term is used to distinguish over analyte transport across thedisrupted surface. The mass transport aspect refers to the physicalmovement of the fluid (as opposed to the movement of energy, or solutes)between body fluids in tissue beneath the target surface and thesurface.

Alternatively, disruption of the target surface can producemicro-pathways that simply allow access to the analyte beneath thesurface from a position on the target surface itself, wherein theanalyte passes to the surface essentially free of net mass fluidtransport. In this regard, the analyte may simply diffuse between thetissue below the target surface and a microenvironment established atthe tissue surface. The term “essentially free” refers to aninsubstantial amount of mass fluid transport between the tissue and thetarget surface.

The term “diffusion” refers to the flux across the disrupted surface(e.g., across disrupted skin tissue) between a body fluid below thesurface and the target surface itself, wherein flux occurs along aconcentration gradient Such diffusion would thus include transport ofthe target analyte to maintain equilibrium between the body fluid andthe target surface. When the concentration of analyte is greater in thebody, analyte diffusion would be toward the target surface. When theconcentration of analyte is greater at the target surface, analytediffusion would be toward the body. In addition, net diffusion ofanalyte from the target surface to the body fluid will occur when theconcentration of analyte in the body decreases with respect to theprevious measurement. Diffusion, however, is not limited to the targetanalyte. Certain means of measurement can generate natural byproducts ofthe analyte. Such byproducts can diffuse from a sensing material incontact with the target surface into the body fluid.

In methods that depend upon such “diffusional” access to the targetanalyte, it is preferred that an interface is applied to disruptedtarget surface to facilitate the establishment and maintenance of anequilibrium concentration of both analyte and any byproducts bydiffusion. In this manner, the methods of the present invention permit avirtually continuous measurement during long-term monitoring withoutsaturating the target surface with byproducts or even the analyteitself. The term “equilibrium” refers to the phenomenon in whichdiffusion has equalized the concentration of analyte on either side ofthe disrupted surface such that there is essentially no concentrationgradient. Diffusion of analyte between the body fluid and the targetsurface allows approach to an equilibrium or steady-state condition.When concentrations of analyte change in the body, a timely dynamicchange in the equilibrium enables continuous monitoring of the analyteconcentration at the tissue surface. The methods of measurement ordetection of the analyte contemplated herein avoid transforming orconsuming a significant amount of the analyte, thereby avoidingsignificant reduction in the amount of analyte at the surface whichcould render it a sink for the analyte. However, even in a situationwhere a sink is created, continuous monitoring of analyte concentrationcan measure the rate of diffusion instead of concentration, for examplein the event that the time to reach equilibrium between the targetsurface and the body fluid is insufficient

After the target surface has been suitably disrupted, access to theanalyte is then available at the target surface. Typically, the analyteis present in a fluid sample that has flowed, exuded or otherwise passedto the surface, substantially instantaneously, or occurring over aperiod of time. Alternatively, no net mass fluid transport occurs, withthe analyte simply diffusing to the target surface. In methods where aparticle injection device is used to disrupt the target surface, thequantity of the analyte that is made available at the target surface maybe varied by altering conditions such as the size and/or density ofsampling particles and the settings of the apparatus used to deliver theparticles. The quantity of fluid released may often be small, such as <1μl that is generally sufficient for detection of the analyte.

Once the analyte is accessible at the target surface, the presenceand/or amount or concentration of the analyte is determined. In thisregard, the target surface is contacted with a suitable sensingapparatus as described herein above. This detection step can be carriedout in a continuous manner. Continual or continuous detection allows formonitoring of target analyte concentration fluctuations.

If desired, a suitable interface material may be applied to the targetsurface to facilitate the detection step. For example, after disruptingthe surface, a gel material can be spread over the target site toprovide an interface material. Examples of particularly suitableinterface materials include a hydrogel, or other hydrophilic polymer,the composition of which is predominantly water for measurement ofwater-soluble target analytes. The hydrogel can be used with or withoutsurfactants or wetting agents. For those methods where diffusionalanalyte-access is-used, the interface material can be formulated toprovide a continuous approach to equilibrium of target analyteconcentration between the interface material and the body fluid. Thephysical properties of the interface material are selected to maintainclose association with the micro-passages or other portals. Examples ofhydrogels include, but are not limited to, a 1% solution of a Carbopol®(B.F. Goodrich Co.; Cleveland, Ohio) in water, or a 4% solution ofNatrosol® (Aqualon Hercules; Wilmington, Del.) in water. In some cases(e.g., diffusional analyte access) it is preferred that the interfacematerial not withdraw a sample of body fluid, nor behave like a sink forthe target analyte. In such embodiments, the composition of theinterface material can be selected to render it isosmotic with the bodyfluid containing the target analyte, such that it does not osmoticallyattract body fluid. Other embodiments can comprise hydrogels including,but not limited to, poly(hydroxyethyl methacrylate) (PHEMA),poly(acrylic acid) (PAA), polyacrylamide (PAAm), poly(vinyl alcohol)(PVA), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA),poly(vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), orpoly(ethylene glycol) (PEG), avoiding polymers that can interfere withanalytical methods for specific target analyte such as normal orchemically modified polysaccharides in the case of glucose measurement.

The composition of the interface material can further be selected torender it isotonic or isosmotic with the body fluid containing thetarget analyte, such that it does not osmotically attract mass flow ofbody fluid. In one embodiment, the composition can comprise a modifiedRinger's-type solution to simulate interstitial fluid having acomposition of NaCl (9 g/l), CaCl₂.2H₂O (0.17 g/l), KCl (0.4 g/l),NaHCO₃ (2.1 g/l), and glucose (10 mg/l). Other embodiments can comprisesimpler or more complex aqueous salt compositions with osmolalityranging from 290 mOsm/kg to 310 mOsm/kg.

The interface material, e.g., the gel, may be applied to the targetsurface as described above and sufficient time allowed for analyte fromthe target surface to equilibrate in the gel prior to the detectionstep. The time may be quite short, such as from 30 seconds to 5 minutes.Detection may then be carried out by contacting the target surface witha reporter apparatus constructed according to the present invention.

The determination step can be generalized as follows. An initial stepcan entail obtaining a raw signal from a sensing device, which signal isrelated to a target analyte present in the biological system. The rawsignal can then be used directly to obtain an answer about the analyte,for example, whether or not the analyte is present, or a directmeasurement indicative of the amount or concentration of the extractedanalyte. The raw signal can also be used indirectly to obtaininformation about the analyte. For example, the raw signal can besubjected to signal processing steps in order to correlate a measurementof the sampled analyte with the concentration of that analyte in thebiological system. Such correlation methodologies are well known tothose skilled in the art.

Alternatively, the sampling (“accessing”) step comprises delivery of aparticulate reporter system (particles that comprise the porous matrixdescribed supra, and which contain a reporter system for detecting theanalyte of interest). As such, the reporter system will penetrate andbecome embedded in the target surface. Here again, the particulatereporter system particles are preferably delivered from a particleinjection device, e.g., a needleless syringe system as described incommonly owned International Publication Nos. WO 94/24263, WO 96104947,WO 96/12513, and WO 96/20022. Delivery of sampling particles from theseneedleless syringe systems is generally practiced with particles havingan approximate size generally ranging from 0.1 to 250 μm, preferablyranging from about 10-70 μm. Particles larger than about 250 μm can alsobe delivered from the devices, with the upper limitation being the pointat which the size of the particles would cause untoward pain and/ordamage to the tissue.

The actual distance to which the delivered particles will penetrate atarget surface depends upon particle size (e.g., the nominal particlediameter assuming a roughly spherical particle geometry), particledensity, the initial velocity at which the particle impacts the surface,and the density and kinematic viscosity of the targeted skin tissue.With appropriate gas pressure, particles having an average diameter of10-70 μm can be readily accelerated through the nozzle at velocitiesapproaching the supersonic speeds of a driving gas flow. Preferably, thepressure used when accelerating the particles will be less than 30 bar,preferably less than 25 bar and most preferably 20 bar or less, and theparticles will be delivered to a substantially uniform and homogenousdepth, e.g., of about 1 to 50 microns below the target surface. It is adistinct advantage of this method that the particulate reporter systemis thus delivered to a homogenous and substantially superficial depth inthe target skin. Both the homogeneity of the particle bed, and thesuperficial depth of the delivered particles enable ready transdermalreadings with a radiation sensing device contacted with the outersurface of the skin, and the superficial delivery further ensures thatthe reporter system is sloughed off with the natural turnover of skincells, typically within about 14-21 days. This is in distinct contrastwith other systems where, for example, a reporter system is “tattooed”into the skin surface using a conventional needle to provide asubstantially permanent and non-homogeneous reporter system bed. Suchtattooed systems pose a safety risk as these foreign components areinserted deep into the skin tissue where they have access to vascularsystems.

Alternatively, the particulate reporter system can be delivered from aparticle-mediated delivery device such as a so-called “gene-gun” typedevice that delivers particles using either a gaseous or electricdischarge. An example of a gaseous discharge device is described in U.S.Pat. No. 5,204,253. An explosive-type device is described in U.S. Pat.No. 4,945,050. One example of a helium discharge-type particleacceleration apparatus is the PowderJect XR® instrument (PowderJectVaccines, Inc., Madison, Wis.), which instrument is described in U.S.Pat. No. 5,120,657. An electric discharge apparatus suitable for useherein is described in U.S. Pat. No. 5,149,655.

A large number of analytes may be detected according to the methods ofthe invention. Suitable analytes include, for example, carbohydrates(e.g., glucose, fructose, and derivatives thereof). As used herein,“carbohydrate” refers to any of the group of organic compounds composedof carbon, hydrogen, and oxygen, including sugars, starches, andcelluloses. Other suitable analytes include glycoproteins (e.g.,glycohemoglobin, thyroglobulin, glycosylated albumin, and glycosylatedapolipoprotein), glycopeptides, and glycolipids (e.g., sphingomyelin andthe ganglioside G_(M2)). Glucose is particularly preferred as an analytedue to its importance in diabetes.

Another group of suitable analytes includes ions. These ions may beinorganic or organic. Examples include calcium, sodium, chlorine,magnesium, potassium, bicarbonate, phosphate, and carbonate. Theinvention is also useful for detecting proteins and peptides (the latterbeing lower molecular weight versions of the former); a number ofphysiological states are known to alter the level of expression ofproteins in blood and other body fluids. Included within this group areenzymes (e.g., enzymes associated with cellular death such as LDH, SGOT,SGPT, and acid and alkaline phosphatases), hormones (e.g., hormonesassociated with ovulation such as luteinizing hormone and folliclestimulating hormone, or hormones associated with pregnancy such as humanchorionic gonadotropin), lipoproteins (e.g., high density, low density,and very low density lipoprotein), and antibodies (e.g., antibodies todiseases such as AIDS, myasthenia gravis, and lupus). Antigens andhaptens are also suitable analytes.

Additionally, the invention is useful for detecting and monitoringanalytes such as steroids (e.g., cholesterol, estrogen, and derivativesthereof). In the case of estrogen, the invention makes it possible tomonitor menopausal patients under estrogen therapy (where estrogenlevels can be quite high). The invention is also useful for detectingand monitoring substances such as theophylline (in asthma patients) andcreatinine (a substance associated with renal failure).

The invention may also be used to detect and monitor pesticides anddrugs. As used herein, “drug” refers to a material which, when ingested,inhaled, absorbed, or otherwise incorporated into the body produces aphysiological response. Included within this group are alcohol,therapeutic drugs (e.g., chemotherapeutic agents such ascyclophosphamide, doxorubicin, vincristine, etoposide, cisplatin, andcarboplatin), narcotics (e.g., cocaine and heroin), and psychoactivedrugs (e.g., LSD).

The invention may also be used to detect and monitor polynucleotides(e.g., DNA and RNA). For example, overall DNA levels may be assayed as ameasure of cell lysis. Alternatively, the invention could be used toassay for expression of specific sequences (e.g., HIV RNA).

As described supra, the invention features in vivo methods for detectingan analyte in an individual. According to this method, the sensingapparatus (containing a fluorescence reagent for detecting the analytethat reversibly binds to the analyte) is placed in communication withthe analyte or with tissue or body fluids of the individual suspected ofcontaining the analyte as described supra. As described supra, thepreferred sensing apparatus is configured to retain the fluorescencereagent while allowing analyte to diffuse into and out of said sensor.The fluorescence reagent may include a specific binding pair, one memberof which is labeled with an energy-absorbing donor molecule (which maybe a fluorophore) and the other of which is labeled with anenergy-absorbing acceptor molecule (which may be a fluorophore). Theexcited state energy level of the donor overlaps with the excited stateenergy level of the acceptor. The sensor is illuminated so as to i)excite the donor or ii) excite both the donor and acceptor. Thefluorescence from the fluorescence reagent associated with the presenceof the analyte is then measured by determining the extent to whichnon-radiative fluorescence resonance energy transfer (“FRET”) occursbetween the donor and the acceptor upon binding. The non-radiativefluorescence resonance energy transfer, in turn, is determined bymeasuring i) the ratio of the fluorescence signal at two emissionwavelengths, one of which is due to donor emission and the other ofwhich is due to acceptor emission, when only the donor is excited, orii) the ratio of the fluorescence signal due to the acceptor followingdonor excitation and the fluorescence signal due to the acceptorfollowing acceptor excitation.

Basic Elements of FRET

FRET generally involves the non-radiative transfer of energy between twofluorophores, one an energy donor (D) and the other an energy acceptor(A). Any appropriately selected donor-acceptor pair can be used,provided that the emission of the donor overlaps with the excitationspectra of the acceptor and both members can absorb light energy at onewavelength and emit light energy of a different wavelength.

The method is described below with particular reference to fluoresceinand rhodamine as the donor-acceptor pair. As used herein, the termfluorescein refers to a class of compounds that includes a variety ofrelated compounds and their derivatives. Similarly, as used herein, theterm rhodamine refers to a class of compounds that includes a variety ofrelated compounds and their derivatives. Other examples ofdonor/acceptor pairs are NBD N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) torhodamine, NBD or fluorescein to eosin or erythrosis, dansyl torhodamine, acridine orange to rhodamine.

Alternatively, both the donor and acceptor can absorb light energy, butonly one of them emits light energy. For example, the donor can befluorescent and the acceptor can be nonfluorescent It is also possibleto make use of a donor-acceptor pair in which the acceptor is notnormally excited at the wavelength used to excite the (fluorescent)donor, however, non-radiative FRET causes acceptor excitation.

Although the donor and the acceptor are referred to herein as a “pair,”the two “members” of the pair can, in fact, be the same substance.Generally, the two members will be different (e.g., fluorescein andrhodamine). It is possible for one molecule (e.g., fluorescein, orrhodamine) to serve as both donor and acceptor, in this case, energytransfer is determined by measuring depolarization of fluorescence.

The concept of FRET is described as follows. The absorbance and emissionspectra of the energy donor, is designated A(D), and B(D), respectively,and the absorbance and emission spectra of acceptor, is designated A(A)and E(A). The absorbance and emission spectra of the donor and acceptormay differ, however, the area of overlap between the donor emission andthe acceptor absorbance spectra is of importance. If, for example,excitation of the energy donor occurs at wavelength L light will-beemitted at wavelength II, the donor's emission wavelength. The acceptor,which normally emits light at wavelength III will not emit any lightbecause the acceptor does not absorb light at wavelength I. However, ifthe donor emission spectra, E(D), overlaps sufficiently with theacceptor absorbance spectra, A(A), a non-radiative energy transferprocess can occur resulting in an emission of light at wavelength III bythe acceptor (A).

The non-radiative transfer process occurs when a donor molecule (D)absorbs the photon with a specific electric field vector, termed E. Inthe excited state the donor molecule will exist as a dipole withpositive charge on one side and negative charge on the other. If anacceptor molecule (A) is sufficiently close to D (e.g., typically lessthan 100 Angstroms), an oppositely charged dipole is induced on theacceptor molecule (it is raised to an excited state). Thisdipole-induced dipole interaction falls off inversely as the sixth powerof donor-acceptor intermolecular distance.

Classically, partial energy transfer can occur. However, this is notwhat occurs in FRET, which is an all or nothing quantum mechanical eventThat is, a donor is not able to give part of its energy to an acceptor.All of the energy must be transferred and energy transfer can occur onlyif the energy levels (i.e., the spectra) overlap. When A leaves itsexcited state, the emitted light is rotated or depolarized with respectto the incident light As a result, FRET manifests itself as a decreasein fluorescence intensity (i.e., decrease in donor emission) atwavelength II, an appearance of fluorescence intensity at wavelength III(i.e., an increase in sensitized emission) and a depolarization of thefluorescence relative to the incident light.

A final manifestation of FRET is in the excited state lifetime.Fluorescence can be seen as an equilibrium process, in which the lengthof time a molecule remains in its excited state is a result ofcompetition between the rate at which it is being driven into this stateby the incident light and the sum of the rates driving it out of thisstate (fluorescence and non-radiative processes). If a furthernon-radiative process, FRET, is added (leaving all else unchanged),decay is favored, which means donor lifetime at wavelength II isshortened.

When two fluorophores whose excitation and emission spectra overlap arein sufficiently close proximity, the excited state energy of the donormolecule is transferred by a resonance induced dipole-dipole interactionto the neighboring acceptor fluorophore. In FRET, a sample or mixture isilluminated at a wavelength which excites the donor but not the acceptormolecule directly. The sample is then monitored at two wavelengths thatof donor emissions and that of acceptor emissions. If donor and acceptorare not in sufficiently close proximity, FRET does not occur andemissions occur only at the donor wavelength. If donor and acceptor arein sufficiently close proximity, FRET occurs. The results of thisinteraction are a decrease in donor lifetime, a quenching of donorfluorescence, an enhancement of acceptor fluorescence intensity, anddepolarization of fluorescence intensity. The efficiency of energytransfer, E_(t), falls off rapidly as the distance between donor andacceptor molecule, R, increases. For an isolated donor/acceptor pair,the efficiency of energy transfer is expressed as:E _(t)=1/[1+(R/R _(o))⁶]  (1)where R is the separation distance between donor and acceptor and R_(o)is the distance for half transfer. R_(o) is a value that depends uponthe overlap integral of the donor emission spectrum and the acceptorexcitation spectrum, the index of refraction, the quantum yield of thedonor, and the orientation of the donor emission and the acceptorabsorbance moments. Forster, T., Z Naturforsch. 4A, 321-327 (1949);Forster, T., Disc. Farada Soc. 27, 7-17 (1959).

Because of its 1/R⁶ dependence, FRET is extremely dependent on moleculardistances and has been dubbed “the spectroscopic ruler.” (Stryer, L, andHaugland, R. P., Proc. Natl. Acad. Sci. USA, 98:719 (1967). For example,the technique has been useful in determining the distances betweendonors and acceptors for both intrinsic and extrinsic fluorophores in avariety of polymers including proteins and nucleic acids. Cardullo etal. demonstrated that the hybridization of two oligodeoxynucleotidescould be monitored using FRET (Cardullo, R., et al., Proc. Natl Acad.Sci., 85:8790-8794 (1988)).

Using the Sensing Apparatus and FRET Reporting Systems to MeasureAnalyte Concentrations

The sensing apparatus and reporting systems of the present invention canbe used to detect a wide range of physiological analyte concentrations.In addition, the method is reliable. Also, because the reactants are notconsumed, the devices are reusable for extended periods. Moreover, thein vivo embodiments are non-invasive or minimally invasive.

In general, the sensing apparatus and the FRET reporting system is usedfor analyte detection is one of two ways. The first is a competitiveassay in which an analogue to the analyte being detected and a ligandcapable of binding to both analogue and analyte are labeled, one with adonor fluorophore and the other with an acceptor fluorophore. Thus, theanalogue may be labeled with donor and the ligand with acceptor, or theanalogue may be labeled with acceptor and the ligand with donor. Whenthe labeled reagents contact analyte, analyte displaces analogue boundto ligand. Because ligand and analogue are no longer close enough toeach other for FRET to occur, the fluorescence signal due to FRETdecreases; the decrease correlates with the concentration of analyte(the correlation can be established in a prior calibration step).

In order to be able to reuse the fluorescence reagents, the bindingbetween analyte and ligand should be reversible under physiologicalconditions. Similarly, the equilibrium binding constants associated withanalyte-ligand binding and analogue-ligand binding should be such thatanalyte can displace analogue. In other words, analogue-ligand bindingshould not be so strong that analyte cannot displace analogue.

This approach is applicable to detection of carbohydrates, steroids,proteins, peptides, antigens, haptens, drugs, pesticides, theophylline,creatinine, and small organic molecules generally. In the case ofcarbohydrates such as glucose and fructose, suitable analogue-ligandcombinations satisfying the above-described selection criteria includethe following combinations: glycoconjugate-lectin, antibody-antigen,receptor-ligand, and enzyme-substrate. For example, in the case ofglucose, the combination of dextran as a glucose analogue (as theglycoconjugate) and concanavalin A (as the lectin) is effective. Todetermine suitable combinations for other sugars, one can select alectin that binds to the sugar and then use that lectin in combinationwith bovine serum albumin covalently labeled with that sugar or ananalogous sugar.

In the case of analytes such as steroids, proteins, and peptides, theappropriate combination would be an analogue to the steroid, protein, orpeptide, and an antibody (or antigen, where the protein or peptide is anantibody) or a receptor for the steroid, protein, or peptide. Forexample, in the case of steroids Haugland, R. P. (1989) MolecularProbes: Handbook of Fluorescent Probes and Research Chemicals, MolecularProbes, Eugene, Oreg., provides information on preparation of suitableanalogues. Using cholesterol as a representative example, derivativescan be prepared either by covalent attachment of a fluorophore (e.g.,NBD or pyrene) to the aliphatic side chain or to a hydroxyl group (e.g.,using anthracene as the fluorophore). For cholesterol, the moleculesthus produced are, respectively,22-(N-(7-nitrobenz-2-oxa-1,3diazol-4-yl)amino-23,24-bisnor-5-cholen-3B-ol;1-pyrenemethyl 3B-hydroxy-22,23-bisnor-5-cholenate; and cholesterylanthracene-9-carboxylate. The steroid can also be conjugated to acarrier protein or other macromolecule that would also be fluorescentlytagged with donor or acceptor. The conjugation would again proceed viaeither the aliphatic side chain or the hydroxyl group.

Similar considerations apply in the case of glycoproteins,glycopeptides, and glycolipids. In the case of glycosylated hemoglobin,FRET between a labeled lectin and the heme itself could be measured(this would manifest itself as a quenching of fluorescence).

The second approach using the sensing apparatus and FRET reportingsystem is to select two ligands that bind to different portions (sites)of an analyte molecule; in addition to being spatially different, theportions may be chemically different as well. This approach isapplicable to detection of antigens, haptens, steroids, proteins,peptides, drugs, pesticides, theophylline, creatinine, and large organicmolecules generally. The ligands could be two antibodies, two cellreceptors, or an antibody and a cell receptor. For example, in the caseof hormones such as HCG, FSH, and LSH the labeled ligands could beantibodies or cell receptors that bind to different portions of thehormone molecule.

One variation of this second approach is to detect antibodies such asanti-DNA antibodies in lupus patients by encapsulating two fluorescentDNA fragments, one labeled with donor and the other with acceptor, andthen measuring FRET (which would occur if the antibody of interest werepresent and crosslinked the labeled fragments).

Another variation involves labeled oligonucleotide probes. As describedin Cardullo, R., et al., Proc. Natl. Acad. Sci., 85:8790-8794 (1988),the hybridization of two oligodeoxynucleotides can be monitored usingFRET in conjunction with such probes. In this way, specific DNAsequences can be determined

To assay overall DNA levels, reagents that bind non-specifically to DNAor RNA are used. Examples of such reagents include fluorescentintercalating dyes that show dramatic spectral shifts upon binding.

In yet another variation, a single material is labeled with both donorand acceptor fluorophores. The fluorescence change associated with theconformational change in the material upon binding to analyte is used asan indication of analyte presence. For example, the analyte may be ahelical DNA molecule and the fluorescence reagent is a material labeledwith donor and acceptor fluorophores that binds to the DNA. Bindingchanges the separation distance between the donor and acceptor, and thusthe signal detected by FRET.

Using the Sensing Apparatus and FRET to Measure Glucose Concentrations

One aspect of the present invention relates to a sensing apparatus and aFRET reporting system in a method of detecting and quantifying glucosein a body fluid. The present method relies on the process ofnon-radiative fluorescence resonance energy transfer (FRET) to determinethe occurrence and extent of binding between members of a specificbinding pair that is competitively decreased by glucose. Members of thebinding pair are a ligand (e.g., a lectin, monoclonal antibody) and acarbohydrate-containing receptor (referred to as a glycoconjugate),which binds specifically to the ligand in competition with glucose. Boththe ligand and the glycoconjugate are fluorescently labeled, buttypically are not labeled with the same fluorophore. They are broughtinto contact with a sample as described supra (e.g., interstitial fluid)in which glucose concentration is to be determined.

The present sensing apparatus and FRET reporting system and method areparticularly useful in the day-today monitoring of glucoseconcentrations in individuals in whom glucose homeostasis is compromised(e.g., diabetic or hypoglycemic individuals) and in biomedical research.

Using the sensing apparatus and RET to measure glucose concentrations inbody fluid is described as follows. One macromolecule of the reportingsystem (designated M) includes a covalently bound fluorophore and isreferred to as a glycoconjugate (e.g., dextran). A second macromoleculeof the reporting system (designated L) includes a ligand that has a highdegree of specificity for glucose (e.g., concanavalin A) and afluorophore that is generally not the same fluorophore as that on thefirst macromolecule.

One of these fluorophores is chosen to be a donor and the other is anacceptor as described previously. For the purposes of this illustration,the donor molecule has been placed on the glycoconjugate and theacceptor has been placed on the ligand. The association can then bediagrammed as:DM+AL→DM−LA,where DM stands for Donor-Macromolecule, AL stands for Acceptor-Ligand,and DM−LA represents the association between the glycoconjugate presentin the first complex and the ligand present in the second complex. Uponassociation, the two macromolecules are now close enough to allow energytransfer between the donor and the acceptor to occur.

Spectra are collected by exciting fluorescein at 472 ηm and scanning theemission from 500-650 ηm. Typically, fluorescence intensities aremonitored at the emission maxima for fluorescein (about 520 ηm) andrhodamine (about 596 ηm). The measure of energy transfer is the ratio offluorescence intensities at 520 ηm and 596 ηm (i.e., FI 520/HI 596) as afunction of glucose concentration or the quenching of fluorescein at 520ηm as measured by a fluorimeter.

The presence of free glucose introduces a competitive inhibitor into theformula because free glucose competes with the conjugated dextran forthe ligand. Thus, increasing concentrations of glucose produces adecrease in the amount of ligand binding to the glycoconjugate. Atrelatively low concentrations of glucose, the transfer efficiency willremain high, since little of the macromolecular association will beaffected. At high concentrations of glucose, the transfer efficiencywill be low, due to the fact that the glucose has successfully competedthe ligand off of the dextran.

The methods of the subject invention can be used to detect and quantifyglucose in samples of a size appropriate for obtaining from anindividual (e.g., 0.1-10 μl).

Based on the methods of the subject invention, a number of reportersystems can be constructed to detect glucose concentration in blood invivo. These reporter systems can remain active for extended periods oftime (e.g., one day or more) before having to be replaced. Typically, anew sensing apparatus containing a reporter system is applied to theskin or mucosal surface on a daily basis.

Accordingly, a novel sensing apparatus and monitoring methods aredisclosed. Although preferred embodiments of the subject invention havebeen described in some detail, it is understood that obvious variationscan be made without departing from the spirit and the scope of theinvention as defined by the appended claims.

1. A sensing apparatus, comprising: (a) a substantially planar occlusivebacking; and (b) a reporter system that absorbs or emits a detectableradiation, said reporter system attached, adhered, or otherwiseconnected to a first planar surface of the occlusive backing, whereinsaid reporter system binds an analyte of interest and the ability ofsaid reporter system to absorb or emit radiation is detectably alteredin a concentration dependent manner when the analyte is bound to saidreporter system.
 2. The apparatus of claim 1 wherein said occlusivebacking has sufficient drape characteristics to allow positioning ofsaid apparatus over a skin or mucosal surface.
 3. The apparatus of claim1 wherein the reporter system comprises a specific binding pair having afirst component that is an analyte-specific binding ligand comprising afirst light-absorbing material, and a second component that binds to thebinding ligand of said first component and comprises a secondlight-absorbing material, wherein: (a) binding of said second componentto the first component is reversible; (b) the analyte binds to the firstcomponent in a competitive manner, thereby displacing said secondcomponent; and (c) displacement of the second component produces adetectable alteration in the energy transfer between the first componentand the second component, wherein said alteration is proportional to theconcentration or amount of said analyte that binds to the firstcomponent.
 4. The apparatus of claim 3 wherein the binding ligand is aglucose binding ligand and the analyte of interest is glucose.
 5. Theapparatus of claim 4 wherein said ligand is concanavalin-A.
 6. Theapparatus of claim 4 wherein the second component comprises a dextranglycoconjugate.
 7. The apparatus of claim 3 wherein the first and secondlight-absorbing materials are fluorophores.
 8. The apparatus of claim 3wherein the detectable alteration in the energy transfer between thefirst component and the second component comprises a non-radiativefluorescence resonance energy transfer between said first and secondlight-absorbing materials.
 9. The apparatus of claim 3 wherein the firstcomponent of the specific binding pair is tetramethylrhodamineisothiocyanate-concanavalin A (“TRITC-ConA”) and the second component ofthe specific binding pair is fluorescein isothiocyanate-dextran(“FITC-dextran”).
 10. The apparatus of claim 1 wherein the reportersystem is disposed within a polymer matrix having a pore size thatallows for ingress and egress of a fluid containing or suspected ofcontaining said analyte of interest.
 11. The apparatus of claim 10wherein said polymer matrix is in particulate form.
 12. The apparatus ofclaim 11 wherein the polymer matrix is in the form of porous particleshaving a size predominantly in the range of 0.1 to 250 μm.
 13. A methodfor detecting the presence or amount of an analyte present beneath atarget skin or mucosal surface of an individual, said method comprising:(a) disrupting the target surface to create one or more passages in thatsurface sufficient to allow said analyte to flow, exude, diffuse orotherwise pass from beneath the target surface to the target surface;(b) placing the sensing apparatus of claim 1 in contact with the targetsurface and allowing the reporter system to contact analyte that haspassed to the target surface; and (c) detecting an alteration in theability of the reporter system to absorb or emit radiation, therebyobtaining a signal indicative of the presence and/or amount of analytepresent beneath the target surface.
 14. The method of claim 13 whereinthe target surface is disrupted by accelerating particles into saidtarget surface.
 15. The method of claim 14 wherein the particles have asize ranging from 0.1-250 μm.
 16. The method of claim 15 wherein theparticles have a size ranging from 10-70 μm.
 17. The method of claim 13wherein the analyte is glucose.
 18. A method for quantifying glucosepresent in a body fluid beneath a target surface, said methodcomprising: (a) accelerating particles into the target surface, whereinacceleration of said particles into the target surface is effective toallow passage of glucose from beneath the target surface to the targetsurface; (b) contacting the glucose present at the target surface with aspecific binding pair comprising a first component which is a glucosebinding ligand containing a first light-absorbing material, and a secondcomponent which is a glycoconjugate containing a second light-absorbingmaterial, the excited state energy level of the first light-absorbingmaterial overlapping with the excited state energy level of the secondlight-absorbing material, said ligand and said glycoconjugate beingchosen such that they reversibly bind to each other thereby allowingglucose present at the target surface to displace said glycoconjugateand competitively bind to said ligand; (c) determining the extent towhich non-radiative fluorescence resonance energy transfer occursbetween the first light-absorbing and the second light-absorbingmaterial in the presence of the glycoconjugate displaced by glucose andthe ligand reversibly bound to glucose; and (d) comparing the result ofstep (c) with the relationship between the extent of non-radiativeenergy transfer between the first light-absorbing material and thesecond light-absorbing material and glucose concentration in the bodyfluid determined in a calibration step.
 19. The method of claim 18,wherein acceleration of said particles into the target surface serves toincrease the permeability of the target surface.
 20. The method of claim18, wherein the particles are accelerated toward the target surfaceusing a needleless syringe device.
 21. The method of claim 18, whereinthe particles are accelerated toward the target surface at a velocity ofabout 100 to 2,500 m/sec.
 22. The method of claim 18, wherein theparticles have a size predominantly in the range of 0.1 to 250 μm. 23.The method of claim 18, wherein the particles penetrate the skin to adepth in the range of 1 to 50 μm.
 24. A method for detecting thepresence or amount of an analyte present beneath a target skin surfaceof an individual, said method comprising: (a) providing a particulatereporter system, wherein said reporter system binds the analyte ofinterest and the ability of said reporter system to absorb or emitradiation is altered in a concentration-dependent manner when saidanalyte is bound to said reporter system, and said particulate reportersystem is comprised of particles having a size ranging from 0.1-250 μm;(b) administering said reporter system into the target skin surface suchthat said particulate reporter system is delivered to a substantiallyuniform and homogenous depth within said skin; (c) allowing the reportersystem to contact the analyte; and (d) detecting an alteration in theability of said reporter system to absorb or emit radiation therebyobtaining a signal indicative of the presence or amount of analytepresent beneath said target skin surface.
 25. The method of claim 24wherein said particulate reporter system is delivered using a needlelesssyringe.
 26. The method of claim 25 wherein said particulate reportersystem is accelerated toward the target skin surface at a velocity ofabout 100 to 2,500 m/s.
 27. The method of claim 25 wherein saidparticulate reporter system is delivered at a depth of about 1-50 μmbeneath said target skin surface.
 28. The method of claim 24 wherein theparticles have a size ranging from 10-70 μm.
 29. The method of claim 24wherein said reporter system comprises a specific binding pair having afirst component that is an analyte-specific binding ligand comprising afirst light-absorbing material, and a second component that binds to thebinding ligand of said first component and comprises a secondlight-absorbing material, wherein: (a) binding of said second componentto the first component is reversible; (b) the analyte binds to the firstcomponent in a competitive manner, thereby displacing said secondcomponent; and (c) displacement of the second component produces adetectable alteration in the displacement of the second component andproduces a detectable alteration in the energy transfer between thefirst component and the second component, wherein said alteration isproportional to the concentration or amount of said analyte that bindsto the first component.
 30. The method of claim 29 wherein the bindingligand is a glucose binding ligand and the analyte of interest isglucose.
 31. The method of claim 29 wherein said ligand isconcanavalin-A.
 32. The method of claim 29 wherein the second componentcomprises a dextran glycoconjugate.
 33. The method of claim 29 whereinthe first and second light-absorbing materials are fluorophores.
 34. Themethod of claim 29 wherein the detectable alteration in the displacementof the second component produces a detectable alteration in the energytransfer between the first component and the second component comprisinga non-radiative fluorescence resonance energy transfer between saidfirst and second light-absorbing materials.